Expression analysis of miRNA hsa‐let7b‐5p in naso‐oropharyngeal swabs of COVID‐19 patients supports its role in regulating ACE2 and DPP4 receptors

Abstract Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) is the novel coronavirus responsible for worldwide coronavirus disease (COVID‐19). We previously observed that Angiotensin‐converting enzyme 2 (ACE2) and Dipeptidyl peptidase‐4 (DPP4) are significantly overexpressed in naso‐oropharyngeal swabs (NPS) of COVID‐19 patients, suggesting their putative functional role in the disease progression. ACE2 and DPP4 overexpression in COVID‐19 patients may be associated to epigenetic mechanism, such as miRNA differential expression. We investigated if hsa‐let7b‐5p, reported to target both ACE2 and DPP4 transcripts, could be involved in the regulation of these genes. We verified that the inhibition and overexpression of hsa‐let7b‐5p matched to a modulation of both ACE2 and DPP4 levels. Then, we observed a statistically significant downregulation (FC = −1.5; p < 0.05) of hsa‐let7b‐5p in the same COVID‐19 and control samples of our previous study. This is the first study that shows hsa‐let7b‐5p low expression in naso‐oropharyngeal swabs of COVID‐19 patients and demonstrates a functional role of this miR in regulating ACE2 and DPP4 levels. These data suggest the involvement of hsa‐let7b‐5p in the regulation of genes necessary for SARS‐CoV‐2 infections and its putative role as a therapeutic target for COVID‐19.

and underlying patient health conditions. 3 In addition, the clinical expression of COVID-19 may also depend on the inter-individual genetic variability that can make subjects more or less susceptible to the onset and progression of viral infection and therapy response. [4][5][6][7][8] SARS-CoV-2 is a single-stranded RNA (ssRNA) virus; following virus attachment and entry into host cells, the viral particle is uncoated and its positive-sense ssRNA genome is released into citosol, where it serves as a matrix for the host translation machinery to produce viral proteins. 9 This replication cycle potentially exposes viral RNA to an antiviral cellular defence arranged by the host's endogenous microRNAs (miRNAs). These miRNAs could directly degrade viral RNA and/or prevent viral protein translation, such as has been reported for the influenza virus. 10 On the other end, to facilitate their infection, some viruses have developed strategies to exploit host miRNAs that regulate immunology tolerance. 11,12 Recently, an in silico approach identified potential host miRNAs that target genes involved in immune signalling pathways activated by SARS-CoV-2 infection. [13][14][15] All these data suggest an important functional role of miRNAs in contrasting and/or taking part to viral infections.
Although miRNAs constitute only 3% of the human genome, they regulate approximately 60%-70% of coding genes, and alterations of their different expression levels are implicated in the development of several diseases, including viral diseases. 16 Moreover, genes coding for miRNAs, as other genes, show genetic inter-individual variability, and different studies have shown that genetic variants in miRNA genes can influence, in some cases, their expression, maturation, and even affinity with their target genes. 17 Therefore, a possible variable that might be considered to influence the high clinical variability of COVID-19 could also be the presence of these genetic variants, such as SNPs (single-nucleotide polymorphisms) in microRNA target sites (MTSs) or miRNA sequences. 18 In the last 2 years, several studies have investigated the potential function of microRNAs as biomarkers or therapeutic targets in COVID-19, focusing primarily on circulating miRNA alterations. 19 Although differences of miRNA expression profiles have been observed between infected and uninfected subjects, 20 at present, little is known about possible miRNA expression changes in human nasopharyngeal tissue following SARS-CoV-2 infection.
Our previous paper described the significant upregulation of Angiotensin-converting enzyme 2 (ACE2) and Dipeptidyl peptidase-4 (DPP4) genes in naso-oropharyngeal swabs (NPS) of COVID-19 patients, thus suggesting that these receptors may play an important and complementary role in virus entry and in the onset and progression of disease. 21 It has been widely reported that ACE2, highly expressed in the lungs and in particular in alveolar type II (AT2) cells, is the host receptor for SARS-CoV-2. 22-24 ACE2 is a type I integral membrane protein, involved in the regulation of the renin-angiotensin system and blood pressure, by the conversion of angiotensin II to the vasodilator Ang- (1)(2)(3)(4)(5)(6)(7). ACE2 is also involved in the regulation of several signalling pathways, including integrin signalling. 25 However, DPP4/CD26 has been reported as a co-receptor of ACE2; in fact, SARS-CoV-2, such as MERS-CoV, interacts with the identical residues of DPP4, in particular K267, R336, R317, and Q344. 26,27 DPP4 is a type II transmembrane glycoprotein, with serine exopeptidase activity, expressed in alveolar epithelial cells, endothelial cells, and in bronchiolar epithelial cells and involved in various biological processes as inflammation with post-translational cleavage of hormones and chemokines, T-cell activation, cell adhesion, and apoptosis. 27 Moreover, DPP4 had different roles in nutrition, metabolism and immune and endocrine systems. 28 Literature data described several putative miRNAs targeting and regulating ACE2 and DPP4 genes. [29][30][31][32][33] Among these, hsa-let7b-5p is a miRNA that potentially could target both ACE2 and DPP4 genes. Indeed, Zhang  Hsa-let7b-5p, one of the most studied members of hsa-let7 miRNA family, has been extensively investigated in several diseases, such as cancer, cardiovascular diseases, 34 and type 2 diabetes mellitus, 35 and it is involved in inflammatory processes. 34,36,37 Noteworthily, hsa-let7b-5p potentially could target SARS-CoV-2 in human bronchial epithelial cells according to a bioinformatics prediction. 38 Moreover, hsa-let7b-5p also emerged among host miRNAs, identified by in silico analysis, that target genes involved in immune response pathways to SARS-CoV-2. 39 ACE2 and DPP4 overexpression, observed in NPS of severe COVID-19 patients, 21 may also be linked to the regulatory effect of hsa-let7b-5p. The aim of this study is to investigate the putative modulation of ACE2 and DPP4 expression by hsa-let7b-5p and analyse its expression level in NPS of COVID-19 patients.

| In silico prediction analysis
We performed an in silico analyses using the MiRWalk database (http://mirwa lk.umm.uni-heide lberg.de/) to identify the putative binding sites of hsa-let7b-5p on DPP4 and ACE2 transcripts.
The MiRWalk database provides the largest available collection of miRNA-target interactions obtained from 12 established prediction programs. This database stores predicted data obtained with a machine-learning algorithm including experimentally verified miRNA-target interactions.

| Cell culture and miRNA overexpression and inhibition
HeLa cell line (ATCC) was cultured in complete medium DMEM

| Expression study on transfected HeLa cells
Total RNA of transfected HeLa cells was extracted using TRIzol reagent (Ambion) according to the manufacturer's instructions.
One μg of total RNA was retrotranscribed in cDNA using the High Capacity cDNA Reverse Transcription Kit (Thermofisher Scientific) according to the manufacturer's instructions. To evaluate the expression level of ACE2 and DPP4, a qRT-PCR (SYBR Green assay Applied Biosystems™) assay was performed, using Real-Time PCR 7500 Software (Applied Biosystems™). Primers, qRT-PCr conditions, and data analysis have been performed as described in our previous paper. 21 GADPH has been selected as the housekeeping gene for data normalization in HeLa cells.
For the hsa-let7b-5p expression analysis, 500 ng of total RNA was retrotranscribed into cDNA using the miScript II RT kit (QIAGEN). To evaluate the hsa-let7b-5p expression level, ABI7500

| Patients' recruitment
Sixty naso-oropharyngeal swabs, derived from a cohort of COVID-19 patients and SARS-CoV-2 negative subjects enrolled from 20th March to 20th April 2020, during the first severe pandemic wave in Italy, were collected to analyse miRNA expression. A venous blood sample (2 ml BD Vacutainer® EDTA Tubes) of all 60 subjects was also collected during their transition to the Tor Vergata University Hospital's, to sequence the LET7b gene.
Available clinical data and SARS-CoV-2 host gene expression levels were extrapolated from previous analysis as already described in our previous paper. 21 This study was approved by the ethical committee of Tor Vergata University Hospital -Rome, Italy (protocol no. 50/20), and all patients received and signed a written informed consent, in agreement with the principles of the Declaration of Helsinki.

| Hsa-let7b-5p expression study in nasooropharyngeal swabs
The residual swabs, obtained after viral RNA detection, contain both human RNA and microRNAs. RNA in residual swabs was evaluated by NanoDrop DS-11 (DeNovix), and to isolate miRNA fraction, 50 ng of total RNA was retrotranscribed into cDNA using the miScript II RT kit (QIAGEN). The qRT-PCR expression analyses were performed as described in paragraphs 2.3.

| Genomic analysis
Genomic DNA of all 60 participants was extracted from 2 ml of peripheral blood samples using standard procedures and the Qiagen blood

| Statistical analysis
Statistical analyses of expression data were performed using GraphPad Prism 6.0 (GraphPad Software) and SPSS program, ver-

| In silico prediction analysis results
In order to confirm the interaction between hsa-let7b-5p and its two putative target genes, we first performed in silico analyses.
The bioinformatic analysis through the prediction tool MiRWalk highlighted putative target sites of hsa-let7b-5p onto DPP4 and ACE2 transcripts. In particular, for the ACE2 gene, we observed five putative binding sites (High Score = 1) in the two main transcripts (NM_021804 and NM_001371415), while for the DPP4 gene, we found one putative binding site (Score = 0.85) ( Table S1).
All these putative binding sites are located on the coding region of ACE2 and DPP4 genes.

| ACE2 and DPP4 modulation by hsa-let7b-5p mimic/inhibitor
To evaluate the regulatory effect of hsa-let7b-5p on the two putative gene targets, we performed in vitro functional assays. Hsa-let7b-5p mimics and inhibitors were used to functionally investigate the role of this miR on ACE2 and DPP4 expression levels.

| Hsa-let7b-5p expression level in SARS-CoV-2 positive and negative naso-oropharyngeal swabs
We analysed the expression level of hsa-let7b-5p miRNA in the 60 naso-oropharyngeal residual swabs described above and previously investigated for ACE2 and DPP4 expression. 21 In this study, we de-

| Genomic analysis
In order to verify if the presence of genetic variants might associate with different hsa-let7b-5p expression levels, we sequenced the

| Correlation analysis
We analysed, by Pearson correlation test, the relationship among All these data bolster the innovative therapeutic approach that uses synthetic miRNAs (or alternatively siRNAs) to target and suppress specific translation of proteins critical for SARS-CoV-2 production and/or infection. 52 For example, recent studies have tested exosomes, inorganic nanoparticles, and lipid moieties containing a mix of miRNAs that bind different regions on SARS-CoV-2 open-reading frame (ORF) and 3'UTRs. 53,54 Since ACE2 appears to be the primary receptor for SARS-CoV-2, several strategies have already been tested to target it, for example, antibodies against ACE2 or pseudoligands to dominate the binding site for SARS-CoV-2. 55 Moreover, in a recent study, a synthetic peptide homologous to the DPP4 receptor has been tested on three-dimensional complex lung organoid structures derived from F I G U R E 1 Effect of hsa-let7b-5p inhibition on ACE2 and DPP4 mRNA levels in HeLa cell line. Expression level of hsa-let7b-5p, ACE2, and DPP4 at three different time point (24,48 and 72 h) after let7b-5p inhibitor treatment. anova test was used for data analysis. Expression data (2-ΔCt) are represented as mean with SD. *p-value < 0.05, **p-value < 0.01, ***pvalue < 0.001 human-induced pluripotent stem cells as immunotherapeutic candidates for COVID-19 treatment. 56 The aim of our pilot-study is to verify if the deregulated expression of ACE2 and DPP4 genes, previously identified in COVID-19 patients, can also be due to epigenetic mechanisms such as differential miRNA expression. Literature data described putative miRNAs that could target and regulate ACE2 and DPP4 genes. [29][30][31][32][33] In particular, we investigated hsa-let7b-5p expression levels, which seems to target both the genes. 30,31 Our in silico analysis predicts the putative interaction of hsa-let7b-5p with both ACE2 and DPP4 mRNA. This analysis indicated DPP4 and ACE2 among hsa-let7b-5p putative target genes; in fact, both transcripts contained binding sites for hsa-let7b-5p (Table S1).
In vitro overexpression or inhibition of hsa-let7b-5p in an epithelial cell line (HeLa) showed a progressive increase in ACE2 and DPP4 mRNA levels after treatment with let7b-5p inhibitor ( Figure 1) and a significant decrease of transcripts levels after let7b-5p mimic treatment ( Figure 2).
Based on these data, we analysed the expression level of hsa-let7b-5p in naso-oropharyngeal residual swabs of the same COVID-19 patients and negative subjects for SARS-CoV-2 infection, analysed in our previous study. 21 We revealed a statistically  To the best of our knowledge, this is the first study that ex- writing -review and editing (equal).

FU N D I N G I N FO R M ATI O N
This study was supported in part by a grant of LazioInnova (Italy, Progetti di Gruppi di Ricerca 2020 A0375-2020-36663 GecoBiomark) to G.N. and A.N.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

I N FO R M E D CO N S E NT
Informed consent was obtained from all subjects involved in the study.

R E FE R E N C E S F I G U R E 3
Hsa-let7b-5p expression level in Negative subjects vs COVID-19 patients (p-value<0.05). The Kolmogorov-Smirnov test was used to analyse the distribution of expression data. The Mann-Whitney test was used for data analysis. Expression data (2 -ΔCt ) are represented as mean with SD.*p < 0.05.